urolithiasis

Pharmacological Evaluation of Antiurolithic Activity of Leaves of Adansonia digitata Linn.

1

SUBMITTED BYMr. Swaroop Singh

SUPERVISORProf. (Dr.) Neelam Balekar

College of Pharmacy, IPS Academy,

Rajendra nagar, A.B. Road, Indore-452012

2010-2011

1. INTRODUCTION

Urolithiasis denotes stones originating anywhere in the urinary tract, including the

kidney and bladder (Pearle & Nakada, 2009).

Overall male to female ratio of 2.4:1 increased from 1977 (1.86:1) to 2006 (2.7:1)

(Knoll et al., 2011).

Recurrence rate (10 to 20% within 1 to 2 years, 35% within 5 years, & 60% within

10 years) (Berner et al., 2011).

Types of kidney stones (Dipiro et al., 1997)

• Calcium stones (CaOx and CaP)- 80%

• Uric acid- 9%

• Struvite-10%

• Others- 1%

College of Pharmacy, IPS Academy 2

Fig. 1. Stones in kidney

1.1. PATHOGENESIS

Urine is a complex fluid that contains a number of minerals, inhibitors and

promoters of crystallization.

Crystallization can not occurs whether promoters and inhibitors are present or

absent, if a state of supersaturation does not exist.


Fig. 2. Scheme of the precess of stone formation (Tsujihata, 2005).

1.2. Possible series of events of the normal and pathological crystallization in urine (adopted from ‘Oxford Textbook of Clinical Nephrology’).


Fig. 3. Normal crystallization in urine.

Fig. 4. Pathological crystallization in urine.

1.3. Risk factor associated with kidney stones

Low urine volume

Hypercalciuria (most common)

Hypocitraturia

Hyperoxaluria


Primary defect in renal tubular

reabsorption of calcium

Renal hypercalciuria

Excretion of Calcium in urineIon activity product

Supersaturation Plasma level of calcium

Resorptive hypercalciuria

Production of vit. D3 in kidney

PTH Calcium transport from lumen of gut

Absorptive hypercalciuria

Fig.5. Types of hypercalciuria

2. MANAGEMENT OF KIDNEY STONES

Therapeutic approaches

Removal of stones (surgical management & MET)

Lowering supersaturation (fluid intake & dietary modification)

Prevent crystal retention in kidneys

Reduction of renal oxidative stress

2.1. SURGICAL MANAGEMENT (Pearle & Nakada, 2009)

1) ESWL

2) PCNL

3) URS (flexible ureteroscopy)


Cont…

2.2. MEDICAL EXPULSIVE THERAPY (Pearle & Nakada , 2009)

A new clinical approach

Urethral smooth muscle relaxation

CCB & alpha-1 receptor blockers

2.3. FLUID INTAKE AND DIETARY MODIFICATION

High fluid intake in order to maintain a urine output of at least 2 L/day

Reduced supersaturation and dilutes promoters of CaOx crystallization

Restriction of animal protein diet

Restriction of Na intake (2-3gm)

No restriction of calcium


Cont…

2.4. PHARMACOLOGICAL TREATMENT (Dipiro et al., 1997)

Diuretics

Alkali Potassium citrate, potassium magnesium citrate

Pyridoxine

2.5. LIMITATION

Persistent of stone fragments cause renal injury & an increase in stone recurrence

with ESWL & development of diabetes and hypertension on long term use

(Butterweck & Khan 2000).

Long term use of thiazide causes K depletion (hypokalemia), Fatigue, dizziness,

impotence & musculoskeletal symtoms.


3. PLANT PROFILE

3.1. BIOLOGICAL SOURCE

Botanical name : Adansonia digitata L.

Family : Bombacaceae

3.2. PHARMACOLOGICAL ACTIVITIES OF LEAVES OF PLANT

Anti-ulcer activity (Karumani et al., 2008)

Antimicrobial activity (Oloyede et al., 2010)

Antioxidant activity (Vetuani et al., 2002)

Antiviral activity (Hudson et al., 2000 ; Ananil et al., 2000)

Diuretic activity (Kirubha et al., 2006)

Anti-inflammatory activity (Selvarani & Hudson, 2009)

Antisickling activity (Adesanya et al., 1988)


3.3. TRADITIONAL USES OF LEAVES OF Adansonia digitata (Sibibe & Willians, 2002)

Insect bite & gunia worm sores & otitis

Diseases of the bladder and urinary tract

Antiasthmatic

Antipyretic

Fatigue

Dysentery

Opthalmia

Astrigent

Diaphoretic

Tonic


Fig.6. Leaf of Adansonia digitata.

3.4. PHYTOCHEMICAL CONSTITUENT OF LEAVES OF PLANT

Excellent source of amino acids (Yazzie et al., 1994)

Carbohydrate , fat, protein and fibre (Caluwe., 2010)

Antioxidants such as catechins and adansonia flavonosides (Maranz et al., 2007;

SEPASAL database., 2007; Kunkel ., 1979)

Minerals (iron, zinc, calcium, magnesium, manganese, copper, phosphorus &

sodium) (Glew et al., 1997)

Provitamin A along with thiamine, riboflavin &niacin (Caluweet al., 2010)

Mucilage (Woolfe et al., 1977)

Alkaloids & Steroids (Oloyede et al., 2010)

Tannis (Oloyede et al., 2010)

Cardio active glycosides (Oloyede et al., 2010)


4. OBJECTIVE

The objective of present study to assess the effectiveness of leaves of Adansonia

digitata L., a medicinal plant used to treat kidney and bladder disease as

traditionally, as a prophylactic agent against CaOx stones by in vitro and in vivo

study.

The present study shall undertake

• Collection of plant material from local area.

• Identification and authentication of the plant from Botanical survey of India.

• Preparation of suitable extracts of selected plant material.

• Evaluation of the extract for antiurolithic activity by in vitro and in vivo study.


4.1. RATIONALE

No satisfactory drug to use in clinical therapy for the prevention or the recurrence

of stones.

Traditional claim of the plant(Sidibe & Willians, 2002; Caius, 2003).

Absence of literatures on the systematic investigation of antiurolithic activity.

Data from In vitro and In vivo & clinical trials reveal that phytotherapeutic agent

could be useful in the management of urolithiasis. In this regards many plant have

been used to treat stones like Dolichous biflorus, Berginia ligulata, Tribulus

terrestris, Orthosiphon grandiflorus, Phylanthus niruri (Butterweck & khan, 2000).

Diuretic activity (Kirubha et al., 2006) & antioxidant activity (Vetuani et al., 2002).

Antiurolithic activity of L-arginine amino acid (Pragasam et al., 2005).


5. PLAN OF WORK


PLAN OF WORK

Phytochemical and pharmacognostic investigations

Collection of plant

Authentication of plant material

Preparation of aqueous extract of leaves

In vitro studyA gel model for measuring crystallization inhibitor activityNucleation and aggregation assay

Detailed pharmacological investigations of antiurilithic activity

In vivo studySodium oxalate induced urolithiasis model in male rats

Minor project Major project

Fig.7. Plan of work

6. EXPERIMENTAL WORKS AND RESULTS

6.1. PLANT MATERIALS AND EXTRACTION PROCEDURE

Leaves of Adansonia digitata were collected from MHOW, Indore & authentication of

plant was done in BSI, Pune (a voucher specimen number BSI/WC/Tech./2011/900A).

Aqueous extract was prepared by decoction method (temperature 90-95ºC) and was dried

at 37ºC. Yild was found 15.8 % (Pauly, 2001).


6.2. AGAR GEL MODEL (Schneider et al., 1983)

Fig.8. Diagram of punch-hole pattern (all dimensions in mm) of agar gel model

6.2.1. Protocols For Agar Gel Model


Group First method Second method

Modifier (70 µL) Modifier (conc. in agar gel)

Control Distilled water Distilled water

Standard Magnesium chloride (100mM) Magnesium chloride (100mM)

Trisodium citrate (100mM) Trisodium citrate (100mM)

Test HA extract (1mg/mL) HA extract (1mg/mL)

HA extract (10mg/mL) HA extract (10mg/mL)

Table 1:Protocols for agar gel model.

6.2.2. Results of Agar Gel Model

College of Pharmacy, IPS Academy 17Fig.9. Pattern of streak of caox cyrstal in agar gel

Crystallization streak’s Pattern

of CaOx crystal produced by

starter solution of 0.3 M CaCl2

and ammonium oxalate in agar

gel with addition of

a. distilled water.

b. 200 mM of MgCl2.

c. 1 mg/mL AAD.

d. 10 mg/mL of AAD.

e. 25 mg/mL of AAD.

f. distilled water.

g. 200 mM of MgCl2.

h. 100 mM of MgCl2.

i. 1 mg/mL of AAD.

j. 10 mg/mL of AAD; n=6

6.3. NUCLEATION AND AGGREGATION ASSAY (Kulaksizoglu et al., 2008 & Hennequin et al.,1993)

The nucleation and aggregation of CaOx were studied at pH 5.7 using

turbidimetric measurment at 620 nm of suspensions produced by mixing calcium

chloride and sodium oxalate (4 mM & 0.5 mM respectively).

Stock solutions (pH 7.5)

• Calcium chloride (8 mM, containing 200 mM NaCl & 10 Mm sodium acetate).

• Sodium oxalate (1 mM, ontaining 200 mM NaCl & 10 Mm sodium acetate).

• Trisodium citrate (0.30 mM) & test solutions (0.2, 2.0 & 20.0) in calcium chloride

stock solution.

All experiments with modifiers of CaOx crystallization were performed at assay

conc. of 4 mM CaCl2, 0.5 mM Na2C2O4, 200 mM NaCl, 10 mM sodium acatate,

0.1, 1.0, 10.0 mg/mL of extract, pH 5.7.



0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

SN Equilibrium SA

Time (min)

OD

(62

0 nm

)

Fig. 10. Time Course Measurements of OD at 620 nm At Standard Condition of CaOx Crystallization

6.3.1. Results of Nucleation and Aggregation Assay.

Parameters Control StdTest

0.1mg/mL 1.0 mg/mL 10.0 mg/mL

tmax (min)7.96 ± 0.31 12.47 ± 0.54 12.38 ± 0.79 13.22 ± 0.38* 23.68 ± 1.69***

SN (×10-2)1.05 ± 0.04 0.36 ± 0.03** 0.52 ± 0.08 0.41 ± 0.02* 0.23 ± 0.04***

% Inhibition of SN - 65.12 ± 3.70 57.71 ± 5.40 60.42 ± 2.20 77.93 ± 4.10

SA (×10-3)1.24 ± 0.05 0.64 ± 0.06** 0.70 ± 0.02 0.57 ± 0.05*** 0.63 ± 0.05*

% Inhibition of SA - 48.42 ± 5.54 43.41 ± 2.04 53.03 ± 4.95 49.19 ± 4.08


Table 2: Effect of extract on calcium oxalate crystallization.

*P < 0.05,** P < 0.01,***P < 0.001 vs control (Kruskal-Wallis variance analysis); n=6-9, ± SEM


Table 3: Treatment Schedule

6.4. ANIMAL MODEL OF SODIUM OXALATE INDUCED UROLITHIATIC IN MALE RATS (Gupta et al., 2006; Khan et al., 1981., Ramesh et al., 2010)

Groups Treatment (daily for7 days)

Normal control 0.9% saline (1 mL/100g, orally & i.p.)

Urolithiactic group NaOx (7 mg/100g, i.p.) + 0.9% saline orally (1 mL/100g)

Standard groupNaOx (7 mg/100g, i.p.) + potassium citrate monohydrate (0.25g/100g, orally)

Treated group NaOx (7 mg/100g, i.p.) + aqueous extract (200mg/kg, orally)

Urine volume, pH, body weight, kidney weight (wet and dry), serum and urine level of creatinine, urea, magnesium, and calcium were evaluated on day 7. In addition, histopathological changes in kidney and oxalate in urine and kidney were evaluated.


Table 4: Effect of ADD on serum chemistry in control and experimental animals

6.4.2. Results of Sodium Oxalate Induced Urolithiasis

Parameters

(mg/dl)Control

Urolithiactic

Control

PCi treated/

Standard group

ADD Treated

group

Ca++ 10.44 ± 2.36 11.99 ± 0.065 10.26 ± 2.68 12.68 ± 4.96

Mg++ 2.48 ± 0.21 1.63 ± 0.21*** 2.76 ± 0.19### 2.90 ± 0.090###

Urea 8.19 ± 1.03 27.36 ± 5.59*** 14.21 ± 4.75### 11.78 ± 1.69###

Creatinine 0.59 ± 0.19 2.13 ± 0.77** 0.89 ± 0.13### 0.85 ± 0.07##

Data are expressed as mean ± SD of 5 animals per group; * p< 0.05, ** p< 0.01, *** p< 0.001

versus control group; # p< 0.05, ## p< 0.01, ### p< 0.001 versus urolithiatic control; one-way

analysis of variance, ANOVA followed by Newman–Keuls test for multiple comparisons


Table 5: Effect of AAD on urinary chemistry in control and experimental animals

Parameters Control group Urolithiactic

control

PCi treated/

standard group

ADD treated

group

Vol. (ml/100 g) 6.34 ± 4.11 4.51 ± 0.48 7.78 ± 1.32 6.02 ± 2.06

pH 8.78 ± 0.24 7.30 ± 0.84* 8.97 ± 0.97# 8.85 ± 0.22#

Ca++ (mg/dl) 28.96 ± 4.99 8.31 ± 1.40*** 4.84 ± 3.47*** 3.35 ± 0.89***

Mg++ (mg/dl) 2.50 ± 0.13 2.09 ± 0.10 2.80 ± 0.17# 3.35 ± 0.43###

Ox (µg/dl) 23.94 ± 0.84 29.48 ± 1.15*** 23.83 ± 0.86### 24.35 ± 0.46###

Urea (mg/dl) 1686 ± 667.7 543.3 ± 174.8* 1529.7 ± 261.5# 1830.1 ± 248.1#

Creatinine

(mg/dl)

57.04 ± 15.05 16.92 ±1.43** 55.27 ±9.44## 41.50 ±4.67#


Table 6: Effect of AAD on kidney weight and chemistry in control and experimental animals

Parameters Control Urolithiactic

Control

PCi treated/

Standard group

ADD Treated

group

Wet Weight (mg/100 g) 385.00 ± 17.65 674.60 ± 62.87*** 474.70 ± 75.02### 527.80 ± 36.19###

Dry Weight (mg/100 g) 104.90 ± 9.46 127.80 ± 9.79** 95.09 ± 12.73## 100.60 ± 13.47##

Width (mm) 7.40 ± 0.89 11.80 ± 0.83*** 9.00 ± 0.70### 9.80 ± 0.83##

length (mm) 13.80 ± 1.30 19.00 ± 0.70** 14.00 ± 3.08## 14.80 ± 2.77##

Ox (µg/100 mg) 3.642 ± 0.22 6.12 ± 1.96* 4.41 ± 0.57# 3.59 ± 0.45#

Ca++ (mg/100 mg) 0.49 ± 0.22 1.34 ± 0.48** 0.75 ± 0.16# 0.76 ± 0.13#


Fig .11. Light microscopic of H & E stained kidney section

Renal tissue of a. Control group’s rats showing normal kidney parenchyma with glomerulus, peroximal and distal convoluted tubules with normal brush-broder and intact glomeruli (×100). b. Control group’s rats showing no sign of crystallization (×400) . c Urolithiatic rats showing focal areas of necrosis, infiltration of inflammatory cells and atrophy glomerulus (×100). d. Urolithiatic rats showing deposition of numerous crystals and presence of casts and extensive haemorrhages in collecting tubules (×400). e. Urolithiatic rats showing deposition of salt of calcium, extensive atrophy of tubular lumen as well as dialated tubules (×400). f. Urolithiatic rats showing extensive haemorrhages and infiltration of inflammatory cells (×400). g. PCi treated rats showed moderate haemorrhage and absence of CaOx crystals (×100). h. PCi treated rats showing regeneration of tubules with large hyperchromic nuclei (×400). i. AAD treated animals showing highly regenerating tubules with normal brush-broder and absence of CaOx crystals (×100). j. AAD treated animals showing no deposition of crystals with rare tissue injury (×400).

7. CONCLUSIONS

7.1. CONCLUSIONS

Extract of leaves of Adansonia digitata could act as antilithic agent , by inhibiting

nucleation and aggregation of calcium oxalate crystals.

High binding of extract with oxalate could prevent dietary absorption of oxalate

form intestine.

In conclusion, based on our present results it seems that AAD contains several

active compounds which in a multifunctional/synergistic approach act as

antiurolithic. The mechanism underlying this effect is mediated possibly through an

antioxidant, antimicrobial, and/or anti-inflammatory activities contained in ADD

and presence of inhibitors of CaOx crystallization like magnesium and acidic amino

acids. In vitro and in vivo experiments confirm that AAD could be beneficial in the

management of CaOx stone disease.


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